Process for preparing lactic-acid products

ABSTRACT

A process for preparing lactic-acid products which involves fermentation of milk or dairy products with live bacteria, viz. the strain Bacillus subtilis 534 deposited at the All-Union Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms, the USSR Academy of Sciences and registered under No. B-1666D; the fermentation is conducted until the desired product is obtained.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the food industry and, moreparticularly, to a process for preparing lactic-acid products.

2. Description of the Related Art

Known and widely employed in the art are processes for preparinglactic-acid products, wherein into a pasteurized milk or buttermilk aleaven containing bacteria producing lactic acid is introduced. Curdlingof the milk or component thereof is effected at a temperature of 28° to38° C. for 10 to 24 hours. After inspection of quality of the resultingproduct (kefir, acidophilus milk) and certification of acceptancethereof, it is ready for sale. One is allowed to store the product at atemperature of 2°-8° C. for 24 hours. In the manufacture of theabove-mentioned products use is made of lactic-acid streptococci,lactic-acid acidophilic and propionic-acid bacteria (SU, A, Nos.1238746, 1287822, 1271479).

The lactic-acid products obtained by the above-mentioned processes havea very slight therapeutic and preventive effect in the case ofdysbacterioses, bacterial infections of the gastro-intestinal tract andare absolutely ineffective in pyo-inflammatory processes, food allergy,or diatheses. Due to a high acidity, there products arecounter-indicated in the case of peptic ulcer and gastritis with anincreased secretion.

In the manufacture of lactic-acid products by the above discussedprocesses contamination of the final product with allegedly pathogenicmicroflora (staphylococci, salmonella, shigella and the like), whichresults in putrefaction of the products and, in some cases, in foodpoisoning, is possible. The known lactic-acid products such as kefir andacidophilus milk have a short storage life (below 24 hours).

SUMMARY OF THE INVENTION

The present invention is directed to the use of a novel species ofbacteria, in a process which makes it possible to prepare lactic-acidproducts exhibiting therapeutic and preventive properties in the case ofbacterial infections, disbacteriosis, diathesis, or food allergy.

This object is accomplished by a process for preparing lactic-acidproducts by fermentation of milk or products thereof with live bacteriawith the formation of the desired product. According the presentinvention as the live bacteria use is made of a strain of bacteriaBacillus subtilis 534 deposited on Mar. 28, 1988, at the All-UnionCollection of microorganisms of the Institute of Biochemistry andPhysiology of Microorganisms, USSR Academy of Sciences, and registeredunder No. B-1666D.

It is advisable that the strain according to the present invention beused in the fermentation process in an amount of from 10³ to 10¹¹ oflive cells per dm³ of milk or dairy products.

The process according to the present invention ensures preparation of aproduct possessing active therapeutic and preventive properties in casesof bacterial infections, disbacteriosis, diathesis, or food allergy.This is due to the fact that the novel strain actively produces, intothe ambient medium, a wide-spectrum antibiotic of the protein nature,proteolytic enzymes, an immunomodulator. Furthermore, the bacteria canpenetrate into the tissue of the pathological focus thus providing atherapeutic effect in the case of pyo-inflammatory processes beyond thegastro-intestinal tract. The prior art dairy products have not been usedfor this purpose as yet.

Owing to a high activity of the antibacterial substance produced by thenovel strain, in the production and storage of lactic-acid productsthere is substantially avoided the risk of growth of pathogenicbacteria: spaphylococci, salmonella, dysentery bacillus, andstreptococci in the dairy products. In the prior art processes thedevelopment of said pathogenic bacteria is encountered rather frequentlywhich results in damage of the product and, in some cases, in poisoningsand outbreak of infection. The dairy products obtained with the use ofthe novel strain can be stored, under similar conditions, for a periodlonger by 2-3 times as compared with the product prepared by the knownprocesses.

The products obtained by the process according to the present inventionhave a new pleasent slightly sweet taste.

BEST MODE FOR CARRYING OUT THE INVENTION

The process according to the present invention is effected in thefollowing manner.

Milk or its products (buttermilk, whey) is pasteurized at a temperatureof from 75° to 100° C. for 15 to 30 minutes.

A leaven is prepared from a lyophilized 3-days' culture of the strainBacillus subtilis 534 which is suspended in sterile milk. The leaven isintroduced into the milk or its products at a temperature of 75°-100° C.at a rate of from 10³ to 10¹¹ live cells per dm³. The fermentation isconducted at a temperature of 30°-38° C. for a period of 6 to 24 hours.After the formation of a uniform dense curd the fermentation iscompleted to give an odourless product with a slightly sweet taste.

The resulting product has its Turner acidity within the range of from20° to 50° T. Phosphase is absent, contamination with foreign microflorais not detected. The dry solids amount ranges from 10 to 14% by mass.

The novel strain Bacillus subtilis 534 employed in the process accordingto the present invention has been isolated from a human being and hasthe following morphological features and physiological properties.

It is bacilliform. The size of cells of a one-day agar culture is(2-4)×(0.6-0.8) μm. Bacteria are virulent. Form spores, do not formcapsules. Gram-positive. Colonies on a meat-peptone agar are rough, withscalloped edges, slightly pink shade, of 2-12 mm diameter. The strain ismultiplied at a temperature of 15°-50° C., optimal growthtemperature--36°-37° C. Cleaves glucose, saccharose, mannitol to an acidwithout evolution of a gas. Does not ferment lactose. Does not formhydrogen sulphide of indole. Evolves acetylmethylcarbinol and catalase.Sensitive to benzylpenicillin, ampicillin, erythromycin, monomycin,linkomycin, tetracycline, insensitive to polymixin.

The strain produces an antibiotic with a wide spectrum of action whichinhibits growth of staphylococci, streptococci, proteum, blue pusbacillus, salmonella, shigella, yeas fungi and the like. Furthermore, itreleases proteolytic enzymes which cleave proteins and animmunomodulator.

The antagonism of this strain in respect of bacterial infections hasbeen studied.

The antagonism of the strain Bacillus subtilis 534 to bacterialinfectants was determined following the procedure suggested by V. I.Nikitenko. Into test-tubes with 5 cm³ of a sterile Kolbach's broth 40-50mln. cells of a test-culture (control) are inoculated and, in the samedoses, together the test-culture and the above-specified strain. Thetest-tubes are subjected to incubation in a thermostat for 24 hours atthe temperature of 37° C. Since the bacteria of the strain Bacillussubtilis 534 only insignificantly changed the density of the broth, thedegree of inhibition of growth of the test-culture was determined on aphotoelectrocolorimeter by the difference between the test broth andcontrol broth respectively. The result was regarded as positive if themixture density was by 1.2 and over times lesser than the density of thetest-culture.

The strain Bacillus subtilis 534 inhibited growth of 34 out of 37studied strains of staphylococcus, 11 out of 12 strains of streptococci,8 out of 12 strains of colibacillus, 7 out of 7 strains of salmonella, 6out of 6 strains of proteus, 7 out of 8 strains of blue pus bacillus.Resistant and less-sensitive were mainly saprophilous strains ofmicroorganisms.

To determine the presence of products of antibacterial substances intothe ambient medium, cultures of the strain Bacillus subtilis 534 (threelaboratory series) are grown for 96 hours in Erlenmeyer's flasks on ashaker (240 r.p.m.) at the temperature of 28° C. on a medium of thefollowing composition, % by weight: peas flour--1.5, saccharose--2.1,starch--0.85, NaNO₃ --0.5, CaCO₃ --0.5, NaCl--0.5, water--the balance.The antibacterial activity of 0.1 cm³ of a filtrate of the culturalliquid is determined by the method of diffusion into a 2% meat-peptoneagar. The size of zones of inhibition of the test-cultures was assessedwith deduction of the recess diameter (8 mm).

The growth of staphylococcus was inhibited in zones of 24-27 mm,colibacillus--18-22 mm, clebsiella--18-21 mm, yeast fungus--22-24 mm.

In the determination of an acute toxicity three series of a preparationof a live culture of the strain Bacillus subtilis 534 are used. Eachseries is introduced intraperitoneally in a single dose to 3 white miceand 3 white rats of the Vistar line in the dose of 10 and 20 bln. cellsper cm³ of a 0.9% solution of sodium chloride respectively. All theanimals survived. They had a good appetite. The animals were slaughteredon the 3-rd, 7-th and 14-th day of the experiment. Histologicalinvestigations revealed no inflammatory or dystrophic changes in thebrains, lungs, kidneys, liver, or heart. In the spleen there was notedan increase in the size of follicles and in the number oflymphohistocytic units in the red pulp.

For the study of the degree of an acute toxicity three series of apreparation from the strain Bacillus subtilis 534 are administered peros with meals to 6 white mice and 6 rats of Vistar line respectivelyonce in the doses of 10 and 20 bln. cells respectively. All the animalssurvived. On the next day they were slaughtered. Histologicalinvestigations of the brains, myocardium, lungs, kidneys, stomach, largeand small intestines have shown no changes as compared to the test groupanimals (10 mice and 10 rats). In the liver there was noted a certainincrease in the number of lymphohistocytic infiltrates along the way ofportal tracts. In the spleen an increase in the size of follicles wasnoticed along with the appearance of a great number of lymphohistocyticunits in the red pulp and of giant pokynuclear cells.

In the determination of chronic toxicity three series of a preparationof the strain Bacillus subtilis 534 were introduced each for 30 daysintraperitoneally to 3 white mice and 3 white rats of Vistar linerespectively in the doses of 200 mln, and 1 bln, cells in 1 cm³ of a0.9% solution of sodium chloride. Furthermore, each of the three seriesof the preparation was administered per os in the same doses to 6 whitemice and 6 white rats of Vistar line. All the animals survived. The massof the animals of the test groups as compared to the control ones (10mice and 10 rats in each) was by 11-17% higher (ρ<0.05). The animalswere slaughtered on the 31-st day. Histological studies of the brains,myocardium, lungs, kidneys, stomach, large and small intestines showedno changes. In the liver lymphohistocystic infiltrates located along theway of portal tracts were encountered in a greater number as compared tothe animals of the control groups. In the spleen of the test groups ofanimals lymphoidization of the red pulp and increased size of follicleswere noted, but no giant polynuclear cells were revealed.

During the experiments for testing a chronic toxicity on the 11-th-21-stday in 6 white mice 23 youngsters were born without malformations. Lateron they gave birth to 7 youngsters without malformations.

For the determination of stability of the strain 9 capsules of apreparation of the strain Bacillus subtilis 534 were stored in tightlyclosed flasks at room temperature for 7 years. Additional 9 capsuleswere stored for 3 months at a temperature of -20° to -22° C., 9 capsuleswere stored at the temperature of 110° C. for 2 months. The number oflive bacteria in the capsules was determined by the method ofinoculation of samples from serial dilutions.

Prior to the beginning of experiments the content of bacteria in acapsule was equal to 5,4±0.5 bln. cells, upon storage for 7years-5.2±0.5 bln. cells, upon storage at -20° to -22° C.-5.4±0.6 bln.cells, at 110° C.-5.3±0.6 bln. cells. Therefore, the data obtained showthat the strain according to the present invention can be stored for 7years within a wide temperature range.

The study of properties of the new strain Bacillus subtilis 534 hasshown that it is non-toxic, does not possess teratogenic and allergeniceffects.

The strain curdles milk and dairy products upon culturing due toevolution of proteolytic enzymes. Furthermore, it liberates anantibacterial substance of the protein nature which has a wide spectrumof action, and an immunomodulator.

The resulting lactic-acid product has a slight sweet taste without anyodour. At a temperature of 2°-8° C. the product can be stored for 7 andmore days without losing its organoleptic and therapeutic-preventiveproperties.

Experiments have been carried out to study the possibility of growth ofa foreign microflora in the manufacture of the lactic-acid product. 3dm³ of milk are pasteurized at the temperature of 90° C. for 30 minutes.The milk is poured into 6 glass bottles of 0.5 dm³ capacity each. Intoeach bottle 1 cm³ of a leaven containing 1 bln of cells of a liveculture of the strain Bacillus subtilis 534 is introduced. The bottlesare closed with cotton-wool-cloth plugs and placed into a thermostat atthe temperature of 37° C. Two hours thereafter into 2 samples 1 bln.cells of a live culture of the strain Staphylococcus aureus areintroduced and into 2 samples 1 bln of cells of a live culture of thestrain Shigella flexneri are introduced into each. The formation of auniform curd took place in the experiments within 14-16 hours, in thecontrol--within 10-12 hours. In the experiments and in the control onlyone strain was inoculated from the dairy product, viz. Bacillus subtilis534.

For a better understanding of the present invention, some specificexamples illustrating the preparation of a lactic-acid product and usethereof are given hereinbelow.

EXAMPLE 1

50 dm³ of milk are pasteurized at the temperature of 75° C. for 30minutes. Right after completion of pasteurization the milk is pouredinto 0.5 dm³ glass bottles into which 1 cm³ of a leaven containing 10bln. of cells of a live culture of the strain Bacillus subtilis 534 areintroduced. The bottles are closed with foil caps and placed into athermostat at the temperature of 30° C. The formation of a uniform densecurd occurs within 6 hours. A product is thus obtained which has aslightly sweet taste, a uniform consistence and no odour; phosphatase isabsent. Acidity is 20° T. There is no contamination with a foreignmicroflora.

The resulting dairy product is stored at the temperature of 8° C. for 7days without losing its organoleptic properties. The acidity hasincreased insignificantly to 28° T. No contamination with a foreignmicroflora is observed. At the same time, under the same conditionsthere have been stored 6 samples of kefir produced with the use oflactic-acid bacteria; the product became putrified within 48 hours.

EXAMPLE 2

2 dm³ of buttermilk are pasteurized for 15 minutes at the temperature of100° C. Immediately thereafter 4 cm³ of a leaven containing 500 cellseach of a live culture of the strain Bacillus subtilis 534 is 1 cm³ areintroduced . The buttermilk with the leaven is placed into a thermostatwith the temperature of 38° C. A complete fermentation occurs after 24hours. A product is thus obtained which has a slightly sweet taste,without odour, acidity -49° T. No contamination with a foreignmicroflora is noticed.

EXAMPLE 3

The lactic-acid product prepared as described in the foregoing Example 1is subjected to testing. Female patient S., 11 months; Diagnosis:diabetes, disbacteriosis. Antibiotics were administered againstpneumonia. Since 6 months meteorism was noted along with eruptions onthe skin of the face and body. Disbacteriosis was found in analysis ofthe stool. The lactic-acid product prepared as in Example 1 wasadministered on an empty stomach 2 times a day in the dose of 10 cm³.The eruptions on the face and skin disappeared on the 7-8-th day. Thestool analysis on the 15-th day showed normalization of the microfloracomposition.

EXAMPLE 4

Used for therapy is the lactic-acid product prepared as in Example 2hereinabove. Patient P., 24 years. Diagnosis: a purulent wound of theright crus, peptic ulcer of the stomach. The product is administered peros twice a day by portions of 200 cm³. After 2 days the strain Bacillussubtilis 534 was found in the wound exudate together with Staphylococcusaureus. The wound has become clean and epithelized on the 11-th day. Thepatient noted a lesser intensity of attacks of hunger pains.

EXAMPLE 5

A lactic-acid product prepared in a manner similar to that described inExample 1 hereinbefore was administered for prophylaxis ofdisbacteriosis in 4 patients in a dose of 10 to 250 cm³ 1-2 times a day;the patients were administered previously with antibiotics for a longtime. Bacterial investigations of the stools made on the 12-th day haveshown that in all of the patients the composition of the microflora doesnot differ from the normal one. Clinical symptoms of bacteriosis werealso absent. At the same time, out of 7 patients of the control group in5 disbacteriosis of various severity was observed.

The dairy products in the same doses were administered for therapy ofdisbacteriosis in 2 patients, diathesis--in 3, purulent wounds--in 3patients (one patient also had peptic ulcer of the stomach). Clinicalarresting of the pathological processes on the 2-nd-9-th day of thetreatment was noticed in all of 8 patients. Bacteriologicalinvestigation of the stool on the 12-th day have shown normalization ofthe microflora composition.

Upon administration of the above-mentioned lactic-acid product to 10healthy volunteers in the dose of 250 cm³ twice a day during one monthno pathological reactions were observed.

INDUSTRIAL APPLICABILITY

The process according to the present invention is useful for themanufacture of food lactic-acid products exhibiting therapeutic andpreventive properties and intended for prophylaxis and treatment ofbacterial infections, disbacteriosis, diathesis and food allergy.

I claim:
 1. A process for preparing lactic-acid products by fermentationof milk or products thereof with live bacteria to give the desiredproduct, which comprises pasteurizing said milk or products thereof;adding to said milk or products thereof, a culture of strain Bacillussubtilis 534; and subjecting said milk or products thereof tofermentation conditions.
 2. A process for preparing lactic-acid productsby fermentation of milk or products thereof with live bacteria to givethe desired product, which comprises pasteurizing said milk or productsthereof for from 15 to 30 minutes at a temperature of between 75° and100° C.; adding to said milk or products thereof an amount of from 10³to 10¹¹ of live cells and spores per dm³ of a culture of strain Bacillussubtilis 534; and fermenting said milk or products thereof at atemperature of from 30° to 38° C. for from 6 to 24 hours.